陈旧动物皮张的古DNA提取方法比较研究

Ming Zhang; Peng Cao; Qing-Yan Dai; Yong-Qiang Wang; Xiao-Tian Feng; Hong-Ru Wang; Hong Wu; Albert Min-Shan Ko; Xiao-Wei Mao; Yi-Chen Liu; Li Yu; Christian Roos; Tilo Nadler; Wen Xiao; E. Andrew Bennett; Qiao-Mei Fu

doi:10.24272/j.issn.2095-8137.2020.377

陈旧动物皮张的古DNA提取方法比较研究

doi: 10.24272/j.issn.2095-8137.2020.377

张明^{1, 2, 3, #},
曹鹏^{1, 2, #},
戴情燕^{1, 2, #},
王永强⁴,
冯晓恬^{1, 2},
汪鸿儒¹,
吴宏⁵,
葛明轩¹,
毛晓伟^{1, 2},
刘逸宸^{1, 2},
于黎⁵,
RoosChristian⁶,
NadlerTilo⁷,
肖文⁸,
BennettE. Andrew^{1, 2, ,},
付巧妹^{1, 2, 3, ,}

1.
中国科学院古脊椎动物与古人类研究所，中国科学院脊椎动物演化与人类起源重点实验室，北京 100044，中国
2.
中国科学院生物演化与环境卓越创新中心，北京 100044，中国
3.
中国科学院大学，北京 100049，中国
4.
新疆文物考古研究所，新疆乌鲁木齐 830011，中国
5.
云南大学云南生物资源保护与利用国家重点实验室，云南昆明650091，中国
6.
Gene Bank of Primates and Primate Genetics Laboratory, German Primate Center, Leibniz Institute for Primate Research，Göttingen 37077, Germany
7.
Wildlife Consultant, Cuc Phuong Commune, Nho Quan, Ninh Binh 430000, Vietnam
8.
大理大学东喜玛拉雅资源与环境研究所，云南大理 671003，中国

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出版历程
- 收稿日期: 2020-12-28
- 录用日期: 2021-03-19
- 网络出版日期: 2021-04-07
- 刊出日期: 2021-05-18

Comparative analysis of DNA extraction protocols for ancient soft tissue museum samples

Ming Zhang^{1, 2, 3, #},
Peng Cao^{1, 2, #},
Qing-Yan Dai^{1, 2, #},
Yong-Qiang Wang⁴,
Xiao-Tian Feng^{1, 2},
Hong-Ru Wang¹,
Hong Wu⁵,
Albert Min-Shan Ko¹,
Xiao-Wei Mao^{1, 2},
Yi-Chen Liu^{1, 2},
Li Yu⁵,
Christian Roos⁶,
Tilo Nadler⁷,
Wen Xiao⁸,
E. Andrew Bennett^{1, 2
, ,},
Qiao-Mei Fu^{1, 2, 3
, ,}

1.
Key Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing 100044, China
2.
CAS Center for Excellence in Life and Paleoenvironment, Beijing 100044, China
3.
University of Chinese Academy of Sciences, Beijing 100049, China
4.
Institute of Cultural Relics and Archaeology in Xinjiang, Urumqi, Xinjiang 830011, China
5.
State Key Laboratory for Conservation and Utilization of Bio-resource in Yunnan, Yunnan University, Kunming, Yunnan 650091, China
6.
Gene Bank of Primates and Primate Genetics Laboratory, German Primate Center, Leibniz Institute for Primate Research, Göttingen 37077, Germany
7.
Wildlife Consultant, Cuc Phuong Commune, Nho Quan, Ninh Binh 430000, Vietnam
8.
Institute of Eastern-Himalaya Biodiversity Research, Dali University, Dali, Yunnan 671003, China

Funds: This work was supported by the Chinese Academy of Sciences (CAS, XDB26000000), National Natural Science Foundation of China (41925009, 41630102, 41672021), CAS (XDA1905010, QYZDB-SSW-DQC003), “Research on the Roots of Chinese Civilization” of Zhengzhou University (XKZDJC202006), Tencent Foundation through the XPLORER PRIZE, and Howard Hughes Medical Institute (55008731)

More Information

Corresponding author: E-mail: eandrew@gmail.com; fuqiaomei@ivpp.ac.cn

#Authors contributed equally to this work

摘要

摘要: 濒危或已灭绝动物的DNA研究通常依据古代或已严重降解的标本遗存。比较成熟的古DNA（ancient DNA，aDNA）提取方法主要针对于骨骼样本，对于皮张和毛发的研究较为少见。与古代的骨骼和牙齿情况相似，陈旧皮张样品中的DNA也由于自然降解等情况的存在，使得获取到的DNA片段长度极短、内源DNA含量极低。因此，针对这些样品，需要开发有效的DNA提取方法。本研究以几十年前到铁器时代的动物皮张和毛发样品为材料，比较2种提取方法（试剂盒法和古DNA实验室法）及相互组合的2种方法的DNA提取效果。研究发现除样品本身的差别（如年代等），皮张样品比毛发样品保留更多的内源DNA。所有方法都能够从陈旧的皮张样品中获取内源DNA，但是实验室的方法在DNA产量和质量上整体优于其它方法，实验室方法在纯化上的表现优于试剂盒方法。
- DNA提取 /
- 古DNA /
- 高通量测序 /
- 陈旧皮张
Abstract: DNA studies of endangered or extinct species often rely on ancient or degraded remains. The majority of ancient DNA (aDNA) extraction protocols focus on skeletal elements, with skin and hair samples rarely explored. Similar to that found in bones and teeth, DNA extracted from historical or ancient skin and fur samples is also extremely fragmented with low endogenous content due to natural degradation processes. Thus, the development of effective DNA extraction methods is required for these materials. Here, we compared the performance of two DNA extraction protocols (commercial and custom laboratory aDNA methods) on hair and skin samples from decades-old museum specimens to Iron Age archaeological material. We found that apart from the impact sample-specific taphonomic and handling history has on the quantity and quality of DNA preservation, skin yielded more endogenous DNA than hair of the samples and protocols tested. While both methods recovered DNA from ancient soft tissue, the laboratory method performed better overall in terms of DNA yield and quality, which was primarily due to the poorer performance of the commercial binding buffer in recovering aDNA.
- DNA extraction method /
- Ancient DNA /
- High-throughput sequencing /
- Historical and ancient skin materials
#Authors contributed equally to this work
注释:

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Figure 1. Experimental design, DNA yield of each sample, and sequence content of library bar plots of this study

A: Experimental design showing different combinations of applied extraction and binding buffers (from kit (K) or laboratory (L)). KK: Both buffers from kit; KL: Lysis buffer from kit, laboratory-made binding buffer; LK: Laboratory-made lysis buffer, binding buffer from kit; LL: Both buffers made in laboratory. B: DNA yield of each sample. (a) Normalized amount of DNA for historical skin samples of monkeys, y-axis shows results of normalized molecule number. (b) Normalized amount of DNA for historical hair samples of monkeys. (c) Normalized amount of DNA for ancient samples of dogs. C: Sequence content of library bar plots (a) DNA yield. (b) Number of uniquely mapped reads. (c) Clonality. Percentage shown as a fraction of highest value among different methods for same samples. Dotted horizontal bar represents average values of each method.

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Table 1. Samples used in this study

Sample	Region, country	Sample age (years before present)	Species
Skin-1	Yunnan Province, China	~30	Rhinopithecus strykeri
Hair-1	Yunnan Province, China	~30	Rhinopithecus strykeri
Skin-2	Yunnan Province, China	~50	Rhinopithecus strykeri
Hair-2	Yunnan Province, China	~50	Rhinopithecus strykeri
Skin-3	Na Hang, Vietnam	Several decades	Rhinopithecus avunculus
Skin-4	Xinjiang Uyghur Autonomous Region, China	~2 400–3 100	Canis lupus familiaris
Skin-5	Xinjiang Uyghur Autonomous Region, China	~2 400–3 100	Canis lupus familiaris
Skin-6	Xinjiang Uyghur Autonomous Region, China	~2 400–3 100	Canis lupus familiaris

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